In recent years, industrial utilization of organic solvents has been producing environmental polution problems by discharge of these compounds or waste waters containing these compounds in many parts of the nation. In particular, soil polution by organic chlorinated compounds is a major social problem, and the technology of repairing the contaminated soil has become more essential. The purification method of the contaminated soil includes physical methods and biological methods.
The physical treatment methods include the air-stripping method (a method of purging air into the contaminated soil which was excavated in order to volatize organic chlorinated compounds contained therein, and of removing them by adsorption on activated charcoal), and vacuum extraction methods (a method in which pipes are driven into the contaminated soil to create the state of reduced pressure so that the organic chlorinated compounds therein are volatized and extracted from the soil). However, these methods require an enormous power for purging the air etc., and have the drawbacks that the former methods require excavation of the soil, while in the latter the extraction efficiency is low and purification does not proceed smoothly under the low concentration of the contaminats. Furthermore, both methods only absorb the contaminating substances to the activated charcoal and therefore require a separate means to detoxicate the contaminating substances.
It has been reported recently that the biological treatment method which is under development utilizes the ability of microorganisms to decompose substances and can completely decompose or detoxicate the contaminating substances, and besides less energy is needed for the treatment as compared with the physical means. Moreover, the biological means permits purification even at low concentrations of contaminants and accordingly expectations on the method are great as a low-cost method for soil purification. The known biological methods include the solid-phase treatment (the excavated soil is mixed with phosphorus, nitrogen, microorganisms etc. to promote decomposition of the contaminating substances by the microorganisms), the slurry treatment method (the excavated soil is mixed with water, phosphorus, nitrogen, microorganisms etc. to treat in the liquid form to promote purification speed of the contaminating substances by the microorganisms), and the on-site treatment method (methane, the air, phosphorus, and nitrogen are injected into the soil without excavating the soil to promote decomposition of the contaminating substances by the microorganisms).
Of the conventionally used biological treatment methods, the solid-phase treatment method and the slurry treatment method require excavation of the soil and besides have a narrow range of application, and the cost for treatment and equipment is relatively high.
On the other hand, the on-site treatment method in which indegenous microorganisms are performed as degraders is less expensive in treatment and equipment compared with the methods described above, and can be applied on a wider range. But, under the condition that the absolute number of microorganisms in the soil is small, the purification rate of the on-site treatment decreases. Especially in the case of the compounds refractory to decomposition such as organic chlorinated compounds, purification is impossible when there are no living microorganisms which can decompose said contaminants in the soil. In such cases, it is believed that inoculation of the microorganisms having the ability of decomposing organic chlorinated compounds into the soil is essential for enhancement of the purification late of soil.
Known microorganisms which decompose trichloroethylene include Methylosinus tricosporium OB3 (Japanese Unexamined Patent Publication (Kohyo) No. 4(1992)-501667, Japanese Unexamined Patent Publication No. 5(1993)-212371), and Methylosinus tricosporium TUKUBA (Japanese Unexamined Patent Publication No. 2(1990)-92274, Japanese Unexamined Patent Publication No. 3(1991)-292970) which are methane-degradatating organisms, Pseudomonas putida F1 (Japanese Unexamined Patent Publication No. 64(1989)-34499), Pseudomonas putida BH (Fujita et al.; Chemical Engineering, 39(6):494-498, 1994), Pseudomonas putida UC-R5, UC-P2 (Japanese Unexamined Patent Publication No. 62(1987)-84780), Pseudomonas putida KWI-9 (Japanese Unexamined Patent Publication No. 6(1994)-70753), Pseudomonas mendocina KR1 (Japanese Unexamined Patent Publication No. 2(1990)-503866, 5(1993)-502593), Pseudomonas cepacia G4 (Japanese Unexamined Patent Publication No. 4(1992)-502277), and Pseudomonas cepacia KK01 (Japanese Unexamined Patent Publication No. 6(1994)-296711) which belong to the genus Pseudomonas, Alcaligenes eutropus JMP134 (A. R. Harker, Appl. Environ. Microbiol., 56(4):1179-1181, 1990), Alcaligenes eutropus KS01 (Japanese Unexamined Patent Publication No. 7(1995)-123976), Nitrosomonas europaea (D. Arciero et al., Biochem. Biophys. Res. Commun., 159(2):640-643, 1989) which is an ammonia-oxidizing bacterium, Corynebacterium J1 (Japanese Unexamined Patent Publication No. 8(1996)-66182) and the like.
The trichloroethylene-decomposing ability of these known microorganisms is not very high and most of these microorganisms can decompose 5 ppm of trichloroethylene in the liquid culture only. Furthermore, since decomposing ability of trichloroethylene in a special environment as the soil is required, it is necessary that the microorganism to be used for bioremediation not only has a sufficient ability of decomposing trichloroethylene but also can remain decomposing ability of trichloroethylene even in the soil. However, most of the known microorganisms are insufficient in this respect.
It is reported that Pseudomonas cepacia KK01 can decompose trichloroethylene at an initial concentration of 30 ppm to 15 ppm in the liquid culture, and trichloroethylene at an initial concentration of 5 ppm to 1 ppm in the soil (Japanese Unexamined Patent Publication No.-6(1994)-296711). Furthermore, it is reported that Alcaligenes eutropus KS01 can decompose trichloroethylene at an initial concentration of 50 ppm to below the level of detection in the liquid culture, and trichloroethylene at an initial concentration of 1 ppm to below the level of detection limit in the soil (Japanese Unexamined Patent Publication No. 7(1995)-123976).
It has been confirmed that these microorganisms have a higher decomposing ability than the conventional microorganisms and that these abilities can be exhibited even in the soil. However, addition of at least one or more than one aromatic compound is needed to the soil environment for induction of the decomposing abilities of these microorganisms. But, the aromatic compounds themselves are contaminants and therefore have a risk of causing a secondary pollution. It is a great challenge to be solved for practical application, therefore, to obtain a microorganism which enables an aromatic compound, when added, to be completely decomposed and removed together with trichloroethylene, or which permits decomposition of trichloroethylene without addition of an aromatic compound.
Accordingly, in order to put the biological purification of trichloroethylene into practical use, it has been desired to obtain a microorganism which has a high decomposing ability, and which enables an aromatic compound, when added, to be completely decomposed and removed together with trichloroethylene, or which permits decomposition of trichloroethylene without addition of an aromatic compound.
Furthermore, in many cases it is extremely difficult to increase the density of a microorganism to the level commensurate with its desired treatment capacity, because the density of the dispersed microorganism is suppressed low in the soil because of predation thereof by protozoa and competitive effects by autochthonous microorganism. In order to increase the density methods are employed such as the method of pressure pumping the air and nutrients into the soil. But despite the enormous energy required, it is difficult to increase the bacterial density by those means alone, thereby keeping the decomposing ability of microorganisms at low levels. Tremendous amounts of energy such as supply of nutrients, aeration etc. are needed to retain a high bacterial density in the closed system such as the reactor as well as in the open system.
If decomposition capability per unit amount of a bacterial mass is increased, a sufficient decomposition capability may be obtained even at low densities of the bacterial mass, thus obviating the need to put in a tremendous amount of energy for keeping the density of the bacterial mass. Though microorganisms which decompose trichloroethylene do so by expressing the enzyme capable of decomposing such substances, such expression of the enzyme requires an inducer. It has already been known that microorganisms can be allowed to exhibit their decomposition ability by adding an inducer and bringing the microorganisms into contact said inducer during culturing, but no previous studies have focused on the length of time of the contact and thereby on the methods to enhance the decomposition ability per unit amount of bacterial mass.
Bio-augmentation which comprises spreading trichloroethylene-decomposing microorganisms into the soil to effect decomposition of trichloroethylene etc. is currently hard to get social acceptance, since it has a potential risk of producing far-reaching effects on the ecological system by releasing a specific microorganism into the environment. But the spraying of a microorganism which has completely lost the propagating activity by sterilization treatment is equivalent to that of mere organic materials, and thus is believed to have little effect on the ecological system. The invention which was disclosed in Japanese post-examined Patent Publication No. 8(1996)-3012 claims that undesirable effects on the ecological system can be minimized by crushing the decomposing bacteria and then spraying them to the soil. But, it will be readily appreciated that the crushing procedure of microorganisms takes extensive equipment, a lot of time and labor, and thus the spraying of a large amount of decomposing bacterium to the contaminated soil will be in fact very difficult.
The above invention further lists the advantageous effects by slaiming that the crushed bacteria are easier to penetrate into the soil than the intact bacterial mass, but said invention makes no mention of duration of the decomposing ability retained by the crushed bacteria. Moreover, the known trichloroethylene-oxidase requires NAD as a coenzyme. But it would be extremely difficult to supply the coenzyme in the concentration necessary for the decomposition reaction of the enzyme which is released from the bacterial mass by crushing the bacterium because the coenzyme is very expensive.